High pCO2 levels are observed in patients with respiratory failure ventilated with the permissive hypercapnia strategy. We and other investigators have recently reported that elevated pCO2 levels can have deleterious effects on the alveolar epithelium. Specifically, we have reported that hypercapnia leads to short and long term dysfunction of the alveolar epithelium by inhibiting the alveolar epithelial Na,K-ATPase and impairing alveolar fluid clearance. Therefore, the focus of this application is to determine the specific mechanisms/signaling pathways that lead to alveolar Na,K-ATPase downregulation and impairment in alveolar fluid clearance during hypercapnic conditions. We propose to study the effects of hypercapnia on the alveolar epithelium via four interrelated aims: in experiments pertaining to specific Aim # 1, we will determine whether the soluble adenylyl cyclase participates in the hypercapnia-induced Na,K- ATPase downregulation in the alveolar epithelium; in experiments pertaining to specific Aim #2, we will determine whether hypercapnia-induced JNK activation leads to LIM-domain only protein 7 (LMO7) phosphorylation and Na,K-ATPase endocytosis; in studies pertaining to specific Aim # 3, we will determine whether Na,K-ATPase ubiquitination leads to its downregulation and propose to identify the specific E2 and E3 ubiquitin ligase; and in studies pertaining to specific Aim # 4, we will determine whether hypercapnia further impairs injury in a model of mild ventilator-induced lung injury (VILI) and whether the downregulation of of sAC, JNK, LMO7 and ubiquitination pathway will reverse the deleterious effects of hypercapnia on alveolar fluid clearance in rodents. In the first cycle of this grant, we have completed most of the proposed research and conducted preliminary experiments for each of the current specific aims which support the feasibility of this grant proposal. The proposed experiments will generate novel information on the effects of hypercapnia on the alveolar epithelium and shed light on the mechanisms leading to alveolar epithelial dysfuntion which is of significance to the understanding and treatment of patients with acute lung injury.